Effects of High Dissolved Inorganic and
Organic Carbon Availability on the Physiology
of the Hard Coral
Acropora millepora
from
the Great Barrier Reef
Friedrich W. Meyer1,2*, Nikolas Vogel2,3, Karen Diele4,5, Andreas Kunzmann1, Sven Uthicke3, Christian Wild1,2
1Department of Ecology, Leibniz Center for Tropical Marine Ecology (ZMT), Bremen, Germany,2Faculty of Biology and Chemistry, University of Bremen, Bremen, Germany,3Australian Institute of Marine Science, Townsville MC, Queensland, Australia,4School of Life, Sport and Social Sciences, Edinburgh Napier University, Edinburgh, United Kingdom,5St Abbs Marine Station, St Abbs, United Kingdom
*friedrich.meyer@hotmail.de
Abstract
Coral reefs are facing major global and local threats due to climate change-induced increases in dissolved inorganic carbon (DIC) and because of land-derived increases in organic and inorganic nutrients. Recent research revealed that high availability of labile dis-solved organic carbon (DOC) negatively affects scleractinian corals. Studies on the inter-play of these factors, however, are lacking, but urgently needed to understand coral reef functioning under present and near future conditions. This experimental study investigated the individual and combined effects of ambient and high DIC (pCO2403μatm/ pHTotal8.2
and 996μatm/pHTotal7.8) and DOC (added as Glucose 0 and 294μmol L-1, background DOC concentration of 83μmol L-1) availability on the physiology (net and gross photosyn-thesis, respiration, dark and light calcification, and growth) of the scleractinian coral Acro-pora milleAcro-pora(Ehrenberg, 1834) from the Great Barrier Reef over a 16 day interval. High DIC availability did not affect photosynthesis, respiration and light calcification, but signifi-cantly reduced dark calcification and growth by 50 and 23%, respectively. High DOC avail-ability reduced net and gross photosynthesis by 51% and 39%, respectively, but did not affect respiration. DOC addition did not influence calcification, but significantly increased growth by 42%. Combination of high DIC and high DOC availability did not affect photosyn-thesis, light calcification, respiration or growth, but significantly decreased dark calcification when compared to both controls and DIC treatments. On the ecosystem level, high DIC con-centrations may lead to reduced accretion and growth of reefs dominated byAcroporathat under elevated DOC concentrations will likely exhibit reduced primary production rates, ulti-mately leading to loss of hard substrate and reef erosion. It is therefore important to consider the potential impacts of elevated DOC and DIC simultaneously to assess real world scenar-ios, as multiple rather than single factors influence key physiological processes in coral reefs.
OPEN ACCESS
Citation:Meyer FW, Vogel N, Diele K, Kunzmann A, Uthicke S, Wild C (2016) Effects of High Dissolved Inorganic and Organic Carbon Availability on the Physiology of the Hard CoralAcropora milleporafrom the Great Barrier Reef. PLoS ONE 11(3): e0149598. doi:10.1371/journal.pone.0149598
Editor:Jiang-Shiou Hwang, National Taiwan Ocean University, TAIWAN
Received:October 27, 2014
Accepted:February 3, 2016
Published:March 9, 2016
Copyright:© 2016 Meyer et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement:All data files are available from FigShare: The DOI of the data is:
https://dx.doi.org/10.6084/m9.figshare.2075254.v1.
Introduction
There is concern about the effects of human-induced increases in atmospheric CO2, which is resulting in increasing dissolved inorganic carbon concentration (DIC) in the world’s oceans. This causes ocean acidification (OA) [1]. The rate of increase of DIC seawater concentration is unprecedented for the last 300 million years [2–5] and will very likely rise further [6] as the partial pressure of CO2(pCO2) is increasing in both atmosphere and the ocean. The resulting reduced pH changes the carbonate system of the seawater by decreasing the saturation state of the different calcium carbonate components [7]. This ultimately affects many coral reef calcify-ing invertebrates such as hard corals, mollusks, echinoderms and foraminifera [8–13] and may lead to changes in calcification, productivity and benthic community structure of coral reefs [14–18]. An increased DIC content can lead to reduced photosynthesis rates of corals [14], increased respiration rates, altered symbiosis and reduced calcification [19–22] even at the lar-val stage [23–26]. In summary, these negative effects can lead to future reef decalcification as shown by mesocosm [27] or field studies [17,28,29].
Recent findings suggest that future predictions ofpCO2levels in seawater are likely a con-servative estimate for highly productive areas such as coral reefs in coastal zones, where large natural variability of the carbonate chemistry [30], coupled with a decrease in buffer capacity, can amplify predicted futurepCO2concentrations up to three fold [31].
While DIC availability directly affects coral reef calcifiers, increased dissolved organic car-bon (DOC) availability indirectly influences corals by altering coral-associated microbial com-munities and stimulating microbial activity [32–35]. However, so far no studies focused on the physiological response of corals towards high DOC concentrations. Thus, scientific knowledge is lacking. Main sources of exogenous DOC are sewage waters [36] and terrestrially derived sediments carrying high amounts of particulate organic carbon that can be transformed into dissolved organic material via microbial degradation [37]. Inorganic nutrients support micro-and macro-algae growth which in turn leads to increased DOC production (up to 1000μmol L
-1, [32]) during bloom and algae mat formation (up to 130
μmol L-1[38]). For the Australian
Great Barrier Reef (GBR), high inputs of inorganic nutrients and increased sediment loads through human activity have led to so called“phase shifts”and changed many coral-dominated to algae-dominated reef communities [39–41].
Nutrient and sediment inputs show high spatial and temporal variation. Land-derived inputs are particularly frequent during the wet season when precipitation is higher, and storm events are recurrent. Nutrient concentrations are particularly high in areas with high river dis-charges [37,42,43]. Both, agricultural land-use and area extent as well as strong storm events and floods likely increase further in the near future [44,45]. Therefore, the contribution of river discharge to DOC availability in the GBR will likely rise, as riverine runoff from agricultural influenced areas is one of the main sources of elevated DOC concentrations. In addition, inor-ganic nutrients may fuel benthic algal growth. Ensuing phase shifts may further increase bio-available DOC production [46,47] and its availability to microbial communities [46,48–53]. These compounds may also promote the growth of pathogens leading to coral bleaching and potential rapid coral mortality [32–34,54,55]. Finally, high microbial activity and degradation of organic carbon reduces the availability of oxygen for the coral holobiont to potentially criti-cal levels [35,38,46,52].
Given the urgent need for understanding coral reef functioning and vulnerability in the Anthropocene, it is surprising that no studies on the combined effects of the important param-eters DOC and DIC have been conducted to date. Studies on the effects on coral physiology, i.e. growth, calcification, and photosynthesis, are lacking, but crucial for evaluating the effects of non-lethal exposure to elevated DOC availabilities. In addition, high DOC availability may Science and Technology for Scotland) and its support
is gratefully acknowledged. MASTS is funded by the Scottish Funding Council [grant number HR09011 to KD] and contributing institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The publication of this article was funded by the Open Access fund of the Leibniz Association.
change the microbial communities associated with the coral holobiont and possibly reduce cal-cification and photosynthetic rates. In combination with high DIC availability, this may result in cumulative negative effects as high DIC may reduce calcification of corals [14,27].
The present study thus investigated the effects of combined high DIC and DOC exposure on a scleractinian coral in a laboratory experiment. We selectedAcropora millepora (Ehren-berg, 1834), a common coral species from the GBR of which the response towards elevated DIC has been studied on the gene expression level [19,56], and effects of elevated DIC on early development and settlement have been described [23]. We monitored photosynthetic perfor-mance as well as growth throughout the experiment over 16 d and measured calcification, oxy-gen and nutrient fluxes as well as chla(chlorophyll a) and protein content at the end of the experiment.
Material and Methods
Specimen collection and preparation
Colonies of the coralAcropora millepora(Ehrenberg, 1834) were collected from reefs next to Pelorus Island (S 18° 33.001’, E 146° 29.304’) in 2012 under a GBMPA sampling permit to the Australian Institute of Marine Science (AIMS). The colonies were fragmented using commer-cial pliers, and individual nubbins (3 to 4 cm height) glued onto ceramic stubs with superglue. Nubbins were mixed from different colonies and maintained in natural seawater flow-through aquaria (volume of several hundred liters) facilities at AIMS under plasma lights (150μmol
photons m-2s-1) in a 12 h/ 12 h light-dark cycle for 3 months to adjust to laboratory conditions and allow to recover from fragmentation until using them for the experiment (see next section).
Experimental setup
Two weeks prior to the onset of the manipulative experiment, 24 nubbins were randomly assigned into 12 experimental tanks (flow-through tanks with 18 l volume each). The experi-ment itself was conducted over a period of 16 d between 24 July and 9 August 2012 at AIMS. Three replicate tanks for the two treatments with two treatment levels were placed in alternat-ing order. The treatments werepCO2in ambient and high availability (403μatm and
996μatm, respectively) and DOC in ambient and high availability (83 ± 10 and
294 ± 506μmol L-1with DOC added as Glucose, D-Glucose, Sigma Aldrich, purity>99,5% in
0 and 377μmol L-1).
DOC & DIC treatment
The high DOC treatment was achieved by daily additions of 1170μmol L-1DOC at 8:00 and
20:00 with pre- weighed Glucose (D-Glucose, Sigma Aldrich), a highly bioavailable short organic carbon molecule. This simulates a sudden increase of DOC and subsequent dissolution as likely to occur in coastal waters, along with sudden increases in river discharges that have been shown to correlate with high amounts of DOC [57–59]. Using stable DOC concentrations would not have adequately reflected natural conditions as production and recycling, especially of high bioavailable DOC occurs on a diurnal basis and even seasonal basis [60]. Dilution by flow through yielded an average concentration of 294 ± 506μmol L-1in the high DOC
treat-ments determined over the seven sample points (n = 2) while background DOC concentrations of the low DOC treatments remained 83 ± 10μmol L-1(Fig 1).
The targetpCO2was 1000μatm, corresponding to levels reached under the Representative
pCO2levels yielded an average of 440μatmpCO2for control and 1090μatmpCO2for high DIC treatments (Table 1). DOC levels were chosen according to maximum concentrations measured in coral reefs (for summary see [32]) and treatments applied in other studies [32,33,54].
Fig 1. DOC concentrations in the high DOC treatment.Time series (08:00 am until 07:00 pm) after addition of 1170μmol L-1DOC as glucose (filled
circles) and a background concentration (unfilled circles) of 76 and 97μmol L-1DOC. Filled circles indicate sampling points (n = 2) for DOC analysis of the
high DOC treatment and unfilled circles of the controls (n = 2 for each point).
doi:10.1371/journal.pone.0149598.g001
Table 1. Carbonate system parameters.
Treatment pH temp Salinity % O2Sat TA pCO2 HCO3- ΩAr
[Total] [°C] [ppt] (μmol kgSW-1) (μatm) (μmol kgSW-1)
Control 8.04 25.4 34.4 105.4 2276.1 402.8 1776.1 4.1
(0.03) (0.2) (0.1) (4.7) (13.0) (10.7) (15.0) (1.2)
High DIC 7.71 25.3 34.4 105.9 2281.2 995.9 2011.4 2.2
(0.04) (0.1) (0.1) (5.7) (7.3) (26.2) (7.6) (0.6)
High DOC 8.02 25.16 34.4 104.8 2286.4 429,8 1791.6 4.0
(0.03) (0.3) (0.1) (6.3) (10.3) (9.9) (8.3) (1.1)
High DIC & 7.69 25.0 34.4 104.2 2281.7 1080.5 2031.7 2.0
High DOC (0.03) (0.4) (0.1) (5.4) (16.9) (15.3) (12.2) (0.6)
Values calculated using CO2Calc with total alkalinity (TA) and pHTotalas input parameters (n = 3 for TA, n = 10 for pH, temperature, salinity and O2).
Values are given as average with standard deviation in parentheses.
Aquaria setup
The corals were kept in freshly filtered (0.5μm) natural seawater at 25°C, with a salinity of
34.5. Water flow was adjusted to 150 mL min-1. Light was provided by individually adjustable white and blue light LED (6000 K, Aqua Illumination). Light levels were set equal to acclima-tion phase. Aquaria pumps (AquaWorld, Australia, 250 L h-1) in each specimen tank provided water movement. Target pH levels were achieved by a pH stat system (Aqua Medic, Germany) controlled by potentiometric pH sensors, as described in Vogel and Uthicke [64]. Total alkalin-ity ATwas determined by gran titration with a Metrohm 855 robotic titrosampler (Metrohm, Switzerland) using 0.5 M HCl (see Uthicke and Fabricius [18]) and certified reference material (CRM Batch 106, A. Dickson, Scripps Oceanographic Institute) for correction. Carbonate sys-tem parameters were calculated with CO2calc software [65] utilizing ATvalues and pH values (Table 1) obtained with a multiprobe (WTW 3430, Germany).
Maximum quantum efficiency
Maximum quantum efficiency (Fv/Fm) was determined by Pulse Amplitude Modulation (PAM) fluorometry using a diving PAM (Walz, Germany) and a 6 mm diameter fiber optic cable. Fv/Fmmeasurements were conducted by light saturation pulse under steady fluoresces signals every evening, after dark adaptation, 30 min after the lights turned off automatically.
Coral surface area
The individual surface area of the incubated coral nubbins from 5 to 16 cm2was determined using the advanced geometry method [66]. Surface areas were calculated as individual col-umns, therefore height and width were measured using Image J software.
Light-/ dark calcification, O
2and nutrient fluxes
After 16 d under experimental conditions, two coral nubbins from each replicate tank were transferred to individual closed plastic chambers (Nalgene 200 mL) and incubated for 60 min in light and 60 min in darkness. PH and DOC concentrations of the seawater in the incubation chambers corresponded to the formerly experienced treatment conditions, and the sea-water was pre-filtered some minutes before the start of each incubation in order to remove bacterial background signals from the incubation water. Each incubation run consisted of 12 parallel incubations in 200 mL closed chambers, including two blanks per treatment. A white light LED (4000 K, Megaman) was installed above each incubation chamber and individually adjusted to meet equal light conditions of the treatment tanks, verified using a quantum sensor (Apogee). To assure constant water temperature during incubation, chambers were placed into a temperature controlled water bath at 25°C, equal to the temperature during the 16 d incuba-tion. Additionally, glass-coated magnetic stirrer bars ensured water movement within the incu-bation chambers.
Light- and dark calcification rates were determined by the alkalinity anomaly technique [67]. A subsample of 50 mL was pipetted from the incubation seawater and directly titrated for total alkalinity measurement by a Metrohm855 (as described above). ATwas calculated by non-linear regression fitting between pH 3.5 and pH 3.0. Calcium carbonate precipitation or dissolution inμM C h-1was calculated by half molar of the difference between the post
incuba-tion and the blank seawater ATreadings, volume of chamber, time of incubation and organism surface area [68].
chamber with fiber optic cables. Net photosynthesis, respiration, and resulting gross photosyn-thesis were determined inμM O2h-1and related to organism surface area. In addition, O2 con-sumption was corrected to blank readings from empty incubation chambers containing only the respective treatment water.
Nutrient fluxes in the chambers were determined by analyzing subsamples of seawater from light and dark incubations for dissolved inorganic nutrients, DIN (NH4+, PO4-and NO2 -+ NO3-as NOx) and total organic carbon, TOC (NPOC) directly subsequent to the experimen-tal runs. Samples for DIN were filtered using 0.45μm syringe filters and kept frozen at -20°C
until measurement by Segmented Flow Analysis (Seal Analytical). Samples for TOC were fil-tered through 0.45μm GFF Filters (Whatman), acidified with 150μL fuming HCl and frozen
at -20°C until analysis on a Shimadzu TOC-5000A (Shimadzu). Nutrient fluxes inμM (DIN)
and mg L-1(TOC) were calculated and corrected for the fluxes of the blank incubations and related to organism surface area.
Growth rates
Coral growth was determined using the buoyant weight technique [69]. Individual specimens were weighed (accuracy: 0.1 mg, Mettler Toledo) in a custom-build buoyant weight set-up with water jacket and seawater of constant temperature (25°C) and salinity (34.5) at the start and end of the experiment. All individuals of all treatments were measured the same day, therefore there was no need of using a standard. Growth of the organisms was expressed as daily percent-age of weight change.
Biological Oxygen Demand (BOD)
To assess effects of elevated organic or inorganic carbon availability on microbial respiration rates, BOD of the treatment water was measured at the end of the experiment for each treat-ment tank (n = 3). For this purpose, 200 mL of unfiltered seawater were incubated for 24 h in the dark under temperature conditions of the treatments. The O2concentration (mg L-1and % saturation) as well as salinity and temperature were recorded before and after the incubation, and O2consumption rates were calculated from these two values and related to water volume and time to mg O2L-1h-1.
Pigment content
Chlacontent ofA.milleporatissue was determined spectrophotometrically. After completion of the incubation experiments, organisms were frozen at -80°C. In the following, the protocol for Chlameasurement described in Vogel and Uthicke [64] and Schmidt et al. [70] was used. Coral tissue was separated from the skeleton by stripping with an air gun using fresh, ultra-fil-tered (0.2μm) seawater. During several subsequent separation steps, the obtained
zooxanthel-lae pellets were kept on ice for further processing, and the host tissue was frozen at -20°C for analysis of total protein content (as described below). Pellets were re-suspended in 5 mL of fresh, filtered seawater, and subsamples of 0.5 mL transferred into 2 mL centrifuge tubes. After centrifuging (10.000 x g for 5 min), the supernatant was discarded, and the zooxanthellae pel-lets were re-suspended in 2 mL of 95% EtOH to extract Chla. Absorbencies were read in 400μL of the supernatant on a 96-well microtiter plate at 750 and 665 nm wavelengths in a
Protein content
Total protein content ofA.milleporawas analyzed with the Bio-Rad protein assay kit (Bio-Rad). Applying the method described in Leuzinger et al. [72], the coral tissue slurry was digested with 1MNaOH for 60 min at 90°C in a sealed deep-well plate. Cell-debris was sepa-rated from the solution (1500 x g for 10 min). Dilutions of protein standard (bovine serum albumin, BSA) and samples were transferred into a 96-well microtiter plate and protein assay reagents were added. After 15 min, absorbency was read on 750 nm wavelength in a Power-wave microplate reader (BioTek). Total protein content ofA.milleporawas calculated, corre-lated to protein standard regression and recorre-lated to nubbin surface area.
Statistical analysis
We tested whether growth rates, light- and dark-calcification rates, photosynthesis, respiration, maximum quantum efficiency, pigment, protein content and nutrient fluxes differed signifi-cantly between treatments and control conditions. Data was tested for normality using the Sha-piro-Wilk test and for equal variance using the Levene median test. Data of net and gross photosynthesis failed the test for equal variance, but showed equal variances after log10 trans-formation. A Two Way ANOVA was then performed with the treatments DIC and DOC as fixed factors to test for treatment effects as well as interactions of treatments and“aquarium”
as nested factor to test for tank effects. To compare differences between individual treatment combinations, a Pairwise Multiple Comparison Procedure (Holm-Sidak method) was per-formed when interactions were significant. All statistical analyses were conducted using Sigma-Plot 12.0 and NCSS statistical statistical software.
Results
Effects of DIC availability
High DIC availability did not affect the BOD of the treatment water compared to controls (Fig 2). It significantly reduced dark calcification rates ofA.milleporaby 50% with 0.06μmol C
cm-2h-1compared to 0.13μmol C cm-2h-1in the control treatments (Fig 3C,S1 Table), but
did not affect calcification in light (Fig 3B). High DIC also reduced growth ofA.milleporaby 23% with 0.2% bw d-1compared to 0.16% bw d-1in the controls (Fig 3A). In contrast, respira-tion rates as well as net and gross photosynthesis were not affected by high DIC (Fig 4A–4C), although photochemical efficiency was significantly reduced by 6% from 0.58 to 0.55 (Fig 5A). Chlaand protein (Fig 5B and 5C) contents ofA.milleporawere not affected by high DIC avail-ability. High DIC availability did however significantly increase NOxuptake of the coral under light conditions by 21% from 0.017 to 0.021μmol cm-2h-1(Fig 6A), while this was not the case
under dark conditions (Fig 6B). Neither NH4+nor PO4-uptake was influenced by high DIC availability under light or dark conditions (Fig 6C–6F). In contrast, DOC release was stimu-lated through high DIC availability by 141% from 1.75 to 0.75μmol cm-2h-1, but only in dark
conditions (Fig 6G and 6H).
Effects of DOC availability
High DOC availability significantly increased BOD by 115% from 0.81 to 1.73 mg L-1h-1 (Fig 2). It did not affect the calcification rate measured during light or dark incubation (Fig 3B and 3C), but increased growth by 42% from 0.20 to 0.29% bw d-1compared to the controls (Fig 3A). The high DOC treatment also reduced net photosynthesis by 51% from 1.24 to 0.63μmol O2cm-2h-1(Fig 4B) and gross photosynthesis by 39% from 1.73 to 1.06μmol
(Fig 4A). Chlaand protein content along with photosynthetic efficiency were unaffected (Fig 5A–5C). The same was observed for NOxfluxes (Fig 6A and 6B). Ammonium uptake however was increased by 36% from 0.007 to 0.009μmol cm-2h-1(Fig 6C) during light conditions, but Fig 2. Biological Oxygen Demand (BOD) in the different treatments at the end of the experiment (n = 3).Data are compared between the control, the high DIC treatment (pCO2403μatm), the high DOC treatment (DOC (added as Glucose 0 and 294μmol L-1) and the combination of both treatments.
Boxplots indicating median (mid of boxplot), 25% and 75% percentile (lower and upper border of boxplot). Significant differences (p<0.05) are marked with an asterisk
doi:10.1371/journal.pone.0149598.g002
Fig 3. Physiological coral responses to treatments.Growth as (a) % change of buoyant weight (BW) ofAcropora millepora(Ehrenberg, 1834) (n = 12). Calcification during light (b) (150μmol photons*m-2s-1) and dark condition (c) measured via alkalinity anomaly technique and related to surface area. Data
are compared between the control, the high DIC treatment (pCO2403μatm), the high DOC treatment (DOC (added as Glucose 0 and 294μmol L-1) and the
combination of both treatments. Boxplots indicating median (mid of boxplot), 25% and 75% percentile (lower and upper border of boxplot) and 90 and 10% percentile (whiskers). Significant differences (p<0.05) relative to controls are marked with an asterisk
not during dark conditions. The latter was also true for PO4-fluxes (Fig 6D–6F). DOC uptake rates were only affected in light incubations and increased by 927% from 0.17 to 1.3μmol cm-2h-1(Fig 6G and 6H).
Combined effects of DIC and DOC availability
The combined high DIC and high DOC treatments led to additive and interactive effects on some of the variables measured. BOD increased further by another 81% compared to the DOC treatment and 197% compared to the controls, with 2.39 mg L-1h-1(Fig 2) indicating a syner-gistic effect.
Coral growth under combined high DOC and DIC availability was similar compared to control conditions, but significantly lower than under high DOC conditions alone and not sig-nificantly higher than under high DIC treatment (Fig 3A). No significant change was observed for light calcification under the combined treatment (Fig 3B). In contrast, dark calcification was reduced by 105% compared to the DIC treatment and 150% compared to the control con-ditions (Fig 3C). High DOC and DIC did not show any combined effects on photosynthesis
Fig 4. Oxygen fluxes as responses to treatments.Net photosynthesis (a), respiration (b) and gross photosynthesis (c) ofAcropora millepora(Ehrenberg, 1834) (n = 6). Net photosynthesis measured during light (150μphotons*m-2s-1) conditions and respiration during dark condition and related to coral surface
area. Data are compared between the control, the high DIC treatment (pCO2403μatm), the high DOC treatment (DOC (added as Glucose 0 and 294μmol
L-1) and the combination of both treatments. Boxplots indicating median (mid of boxplot), 25% and 75% percentile (lower and upper border of boxplot) and 90
and 10% percentile (whiskers). Significant differences (p<0.05) are marked with an asterisk
doi:10.1371/journal.pone.0149598.g004
Fig 5. Photosystem parameters as response to treatments.Maximum quantum yield (a) of dark adapted individuals ofAcropora millepora(Ehrenberg, 1834) (n = 12). Chlorophyll content (b) related to fresh weight (n = 6). Protein content (c) ofA.milleporarelated to surface area (n = 6). Data are compared between the control, the high DIC treatment (pCO2403μatm), the high DOC treatment (DOC (added as Glucose 0 and 294μmol L-1) and the combination of
both treatments. Boxplots indicating median (mid of boxplot), 25% and 75% percentile (lower and upper border of boxplot) and 90 and 10% percentile (whiskers). Significant differences (p<0.05) are marked with an asterisk
Fig 6. Nutrient fluxes as response to treatments.NOxfluxes (a, b), NH4+fluxes (c, d) PO4-fluxes (e, f) and DOC fluxes (g, h) calculated from light
(150μmol photons*m-2s-1) (graphs on the left) and dark incubations (graphs on the right) ofAcropora millepora(Ehrenberg, 1834) (n = 12). Data are
and respiration (Fig 4A–4C). The same was observed for photosynthetic efficiency, Chla con-tent and protein concon-tent (Fig 5A–5C). The uptake rates of NOxin the combined treatment compared to the controls and the DIC treatment were increased in light by 65% and 86%, respectively (Fig 6A). In the dark however, no significant differences were observed (Fig 6B). For NH4+uptake rates, similar results were observed in both dark and light incubations (Fig
6C and 6D). In light, NH4+uptake rates increased by 330% compared to the DOC treatment and 366% compared to the control. In the dark, NH4+uptake rates increased by 75% compared to the DIC treatment and 100% compared to the controls. No such trend was observed for the uptake rates of PO4-(Fig 6E and 6F). However, in light the uptake rates of DOC in the com-bined treatment were increased by 1163% compared to the DOC treatment and by 2090% compared to the controls (Fig 6G). In the dark incubations, DOC uptake rates were not affected by high DOC and DIC concentrations (Fig 6H).
Discussion
The effects of high DIC availability
The BOD in the incubation chambers, an indicator of bacterial respiration during the experi-ment, did not change under high DIC conditions (Table 2). This is consistent with a study on microbial biofilms from the GBR that also reported constant bacterial background respiration and nutrient fluxes, despite different DIC levels [73]. Hence, bacterial communities either did not change during exposure to elevated DIC or rapidly acclimated as suggested by Witt et al. [73]. High DIC did not affect light calcification and photosynthesis ofA.millepora, but decreased dark calcification and growth. This contrasts with a previous study on an acroporid coral that reported a reduction of photosynthetic productivity under elevated DIC conditions [14]. However, in the before mentioned study, high light dosages of up to 1200μmol photons
m-2s-1were used that induced bleaching and consecutively productivity loss. In the present experiment, the continuous light dose of 150μmol photons m-2s-1was comparable to studies
under natural light regimes that found no effect of elevated DIC on the productivity of corals [74,75]. We observed carbonate dissolution under dark incubations (Table 2), which corrobo-rates previous studies that demonstrated negative effects of elevated DIC on coral growth and a net dissolution under similar highpCO2levels [9,14,28]. ThepCO2levels of the high DIC treat-ment corresponded to set treattreat-ment conditions as aimed for [63], and control conditions were close to present-day levels [76] or below levels observed in inshore reefs with higher natural variability [30], but inA.milleporadissolution occurred mainly under dark conditions. combination of both treatments. Boxplots indicating median (mid of boxplot), 25% and 75% percentile (lower and upper border of boxplot) and 90 and 10% percentile (whiskers). Significant differences (p<0.05) are marked with an asterisk
doi:10.1371/journal.pone.0149598.g006
Table 2. Summary of main effects.
Treatment Photosynthesis Respiration Calcification Growth
net gross light dark
High DIC No effect No effect No effect No effect Reduced (50%) Reduced (23%)
High DOC Reduced (51%) Reduced (39%) No effect No effect No effect Reduced (42%)
High DIC & No effect No effect No effect No effect Reduced (50%) No effect
High DOC
Effects as revealed by mixed model ANOVA for photosynthesis, respiration, calcification, and growth are summarized by treatments. Relative values (%) compared to control conditions are given for significant effects.
We show that the effect of ocean acidification on calcification becomes most visible when no photosynthetic activity was present in dark conditions. This supports recent findings that show that respiratory processes may enhance the negative effects of elevated DIC concentra-tions and are the main cause of reduced growth [31]. We found no change in Chlaand protein content which explains the stable productivity under elevated DIC concentrations. However, we recorded a significant reduction in photochemical efficiency, eventually also leading to reduced photosynthetic rates. During the 16 d experiment, we could detect the decline of pho-tochemical efficiency, but no acclimation. Long-term experiments could reveal the cause of a reduced productivity and evaluate potential acclimation.
To our knowledge, the present study is the first to reveal that high DIC stimulates the uptake of NOxand the release of DOC of corals. The uptake rate of NOxunder control conditions lies within described uptake rates for the genusAcropora, although nitrate concentrations were slightly elevated (1.3μM) compared to other studies (e.g. Bythell 1990, 0.22–1.72μM). The
induced NOxuptake probably resulted from a higher demand for nitrogen to allow keeping productivity stable and on a high level, despite reduced photochemical efficiency. The observed increased DOC release on the other hand may have been caused by high availability of bicar-bonate for photosynthesis ensuing increased carbon release when nitrate uptake rates were lower and nitrate becomes limiting [77].
The effects of high DOC availability
In contrast to the high DIC treatment, BOD values in the incubation chambers increased under high DOC. This is in line with other experimental studies investigating the responses of micro-organisms to elevated DOC [32,33,46]. While high DOC reduced photosynthesis ofA.
millepora, it did not affect coral light or dark calcification, but significantly increased coral net growth (Table 2). The reduced photosynthesis rates are likely caused by the high microbial res-piration of the coral host with reduced pH of the water directly on the coral surface compared to the surrounding water. The observed increased net growth of the coral under high DOC availability probably originated from heterotrophic compensation of losses in assimilates due to reduced photosynthesis and the surplus of bio-available organic carbon as energy source. This is supported by the finding that bleached corals can survive through increased heterotro-phic feeding [78,79] and are able to maintain photosynthetic quantum yield during thermal stress [80]. Bleached corals can restore dark calcification when glycerol is added [81], and unbleached corals showed increased calcification rates under glucose addition [82].
The utilization of both ammonia and DOC during light hours under elevated DOC avail-ability may indicate increased microbial activity via ammonia oxidation and carbohydrate metabolism, rather than direct uptake by the coral host itself as supported by the significantly increased BOD. In contrast to other studies, no signs of disease or bleaching occurred during the present study. Although the levels of DOC applied were increased by 500% on average, they were low compared to other studies [32,33,54]. The pH under the high DOC concentra-tion was low, indicating higher bacterial activity as confirmed by a reducconcentra-tion in oxygen satura-tion, the increase in bicarbonate ions and consequently a reduction of the aragonite saturation state.
DOC concentrations described for different reef settings in high DOC environments [32]. In the present experimental study, we used glucose to increase DOC to assure reproducibility. Future studies should now evaluate the effects of combined sugars or different algal exudates in combination with ocean acidification. The latter approach however would need to consider that concentrations of algae- derived DOC change under different light conditions and are dependent on the species of algae [53,60].
The combined effects of high DIC and DOC availability
The combination of both high DIC and DOC availability in this study led to a higher BOD compared to the DOC treatment effect alone (Table 2). This synergistic effect [84] was proba-bly caused by altered, heterotrophic bacterial communities due to a higher stress reaction of the coral towards high DIC and DOC, as described for elevated DIC [85] or DOC availability alone [32]. The combination of both factors significantly decreased dark calcification, increased ammonia, NOxand DOC uptake rates, but did not affect photosynthesis, light calcification or growth. The present study revealed that high DIC and DOC availability has additive negative effects, and the dark calcification was further reduced under the combined treatment than at high DIC levels alone. This may be due to the elevated bacterial respiration under high DOC conditions, which increased the DIC concentration locally above the level of the DIC treatment condition. Hence, the negative impact of the DIC treatment alone was potentially further enhanced due to respiratory processes as mentioned by other studies on DIC effects alone [31]. Overall, growth was similar to the control conditions and likely affected by the surplus of energy for calcification from the DOC treatment, thereby balancing the DIC treatment effect.
The increase in uptake rates of ammonia, NOxand DOC under the combined treatment compared to the individual treatments and the control was likely caused by higher bacterial activity, ammonia oxidation, and nitrate uptake. However, it may also be a sign of shifts in microbial community structure due to excess energy derived from high DOC availability. Additionally, the combined effects of both treatments on the coral host may disrupt‘natural’
bacterial-host interactions. Further studies should now include longer experimental periods as well as stepwise or gradual increase of exposure to both treatments.
Ecological perspective
Our experiment is the first to show that high DOC availability positively impacted coral growth. On the other hand, this study reveals that high DIC negatively affects coral carbonate production because of dissolution at night. The high DIC/DOC combination treatment ampli-fied this negative effect, leading to further decreased carbonate production. Thus, the results of the present study strongly suggest that the simultaneous occurrence of high DIC and DOC in present coastal waters, and likewise even more in future waters, constitutes a serious threat to corals. This will likely negatively influence their ecological functions and services, e.g. habitat provisioning and coastal protection.
water management needs to be taken into account when mitigating potential effects of riverine inputs on coral reefs.
Supporting Information
S1 Table. Results of Two Way ANOVA for DIC and DOC as fixed factors and“aquarium” as nested factor.Degree of freedom (DF), sum of squares (SS) and mean square (MS) as well as F and P values are given. Data for gross and net photosynthesis were log10 transformed prior to analysis. Significant results are marked bold with an asterisk ().
(DOCX)
Acknowledgments
The authors would like to thank the SeaSim team (especially Craig Humphrey) at AIMS for the provision of the coral nubbins. Furthermore, we would like to thank Jane Wu Won at AIMS for the support with the analysis of the TOC samples. In addition, we thank Florita Flores (gen-eral assistance) und Michelle Liddy (field + lab assistance).
Author Contributions
Conceived and designed the experiments: FM NV KD AK SU CW. Performed the experiments: FM NV SU. Analyzed the data: FM NV SU. Contributed reagents/materials/analysis tools: CW SU AK. Wrote the paper: FM NV KD AK SU CW.
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